Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection that has now been well documented in the Northeastern and upper Midwestern United States, and in Europe. The causative agent (recently renamed Anaplasma phagocytophila) is an obligate intracellular pathogen that persists within neutrophils. One of the major functions of neutrophils is to eradicate microbes, and the respiratory burst - initiated by NADPH oxidase - is a major antimicrobial defense mechanism. The goal of this proposal is to understand the pathogenesis of HGE - using in vitro and in vivo models. In particular, our recently published report demonstrates that A. phagocytophila inhibits the respiratory burst by selectively down-regulating gp91phox, a major subunit of the NADPH oxidase holoenzyme. We will now explore this finding in detail. (a) First, we will characterize the respiratory burst during A. phagocytophila infection in vitro, using selected promyelocytic cell lines (HL-60, 32D and MPRO), and human neutrophils, and in vivo using a murine model of HGE and clinical samples from patients with HGE. (b) Secondly, we will examine the specific role of gp91 phox down-regulation by A. phagocytophila in the prevention of NADPH oxidase activity and begin to delineate the mechanism(s) of gp91 phox repression by A. phagocytophila. Our preliminary data strongly suggest that A. phagocytophila infection delays the release of CCAAT-displacement-protein (a potent repressor of gp91 phox transcription) from the gp91 phox promoter, and we will examine this further. Finally (c) we also believe that transcriptional inhibition of gp91phox cannot fully account for complete inhibition of the respiratory burst (in part because neutrophils have some preformed GP91phox protein on the cell membrane). Therefore we will also explore additional mechanisms by which A. phagocytophila influences formation of the active NADPH oxidase complex. These studies should lead to a greater understanding of HGE, an important new tick-borne disease, and the mechanisms that pathogens may use to interfere with neutrophil function.